Investigation of myo-inositol Phosphates on Human Cells by Phenotypic Cell-Based Assays

Abstract

Myo-inositol phosphates (IPs) are a large family of compounds that are ubiquitous in eukaryotes. They are involved in many critical cellular functions, from signal transduction to DNA repair in eukaryotes. Myo-inositol hexakisphosphate (InsP6), commonly known as phytic acid, is the most abundant cellular IP and previous studies have suggested that exogenous InsP6 has anti-proliferative effects on multiple cancer cell lines. By contrast, derivatives of InsP6 with lower phosphate number, such as myo-inositol (1,2,4,5,6)-pentakisphosphate (InsP5), have not been studied due to their costly preparation. Here we investigated the effects of InsP6 and InsP5 on the human bone osteosarcoma cell line, U2OS, by phenotypic assays. After 48 h after treatment with either InsP6 and InsP5 at 15 or 50 µM, cells display a striking vacuole formation, as observed by light microscopy. By staining with acridine orange and observing by fluorescence microscopy, the vacuolated cells showed acidic vesicle accumulation, indicative of increased autophagy. By staining with the non-permeant fluorescent dye Lucifer yellow and fluorescence microscopy we observed that vacuolated cells showed higher levels of the fluorescent dye, indicative of endocytosis, than non-treated cells. Vacuoles observed by phase contrast microscopy are frequently stained with Lucifer yellow and acridine orange. These findings suggest that InsP5 and InsP6 affect endocytosis, which subsequently leads to increased lysosomal degradation the engulfed material. Understanding the effects of IPs on human cells is important in understanding inositol signalling pathways and may lead to the discovery of novel anticancer compounds.

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